多重PCR检测四种食源性病原弧菌

doi:10.3864/j.issn.0578-1752.2013.08.018

中国农业科学 ›› 2013, Vol. 46 ›› Issue (8): 1682-1686.doi: 10.3864/j.issn.0578-1752.2013.08.018

多重PCR检测四种食源性病原弧菌

魏霜, 冼钰茵, 赵晖, 吴希阳

1.暨南大学理工学院食品科学与工程系，广州 510632
2.湖北省出入境检验检疫局检验检疫技术中心，武汉 430022
3.广东省食品安全卫生应急技术研究中心，广州 510632

收稿日期:2012-10-26 出版日期:2013-04-15 发布日期:2013-01-16
通讯作者: 通信作者吴希阳，E-mail：tkentwu@jnu.edu.cn
作者简介:魏霜，E-mail：weishuang2008@hotmail.com
基金资助:
公益性行业科研专项项目（201110039）

Simultaneous Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae Using Multiplex PCR

WEI Shuang, XIAN Yu-Yin, ZHAO Hui, WU Xi-Yang

1.Department of Food Science and Engineering, Ji’nan University, Guangzhou 510632
2.Hubei Entry-Exit Inspection and Quarantine Bureau Inspection and Quarantine Technology Center, Wuhan 430022
3.Food Safety and Health Emergency Technical Research Center of Guangdong Province, Guangzhou 510632

Received:2012-10-26 Online:2013-04-15 Published:2013-01-16

摘要/Abstract

摘要： 【目的】建立在扩增内标存在下同时快速检测溶藻弧菌、副溶血弧菌、创伤弧菌、霍乱弧菌4种食源性致病菌的五重PCR方法。【方法】以细菌16S rRNA基因为扩增内标靶序列，针对溶藻弧菌gyrB基因、副溶血弧菌collagenase基因、创伤弧菌vvhA基因、霍乱弧菌ompW基因，分别设计特异性引物，建立多重PCR体系，对其灵敏度和特异性进行评价，并将建立的五重PCR应用于弧菌的筛选。【结果】建立的多重PCR检测体系对混合模板中副溶血弧菌、溶藻弧菌、创伤弧菌的灵敏度达10 CFU/mL，霍乱弧菌的灵敏度达105 CFU/mL，经特异性评价证实其特异性好，并能有效指示PCR反应的假阴性，将建立的多重PCR应用于69株疑似弧菌菌株的鉴定，其结果与生理生化鉴定结果一致。【结论】该方法能够快速、灵敏、准确地检测溶藻弧菌、副溶血弧菌、创伤弧菌和霍乱弧菌4种食源性致病菌，灵敏度高，并能有效指示PCR反应的假阴性，适用于食品中常见致病性弧菌的快速筛检。

关键词: 弧菌 , 检测 , 多重PCR

Abstract: 【Objective】The objective of this study is to develop a multiplex PCR assay that can simultaneously detect Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in the presence of an internal amplification control (IAC).【Method】Species-specific PCR primers were designed based on gyrB gene for Vibrio alginolyticus, collagenase gene for Vibrio parahaemolyticus, vvhA gene for Vibrio vulnificus and ompW gene for Vibrio cholerae, 16S rRNA gene of bacteria as IAC primers was used to indicate false-negative results. Multiple PCR method was developed after optimization reaction condition. 【Result】The multiple PCR method was proved to be rapid, high-throughput, sensitive and specific and the existence of IAC could successfully eliminate false-negative results. The multiplex PCR was validated with 69 suspicious Vibrio strains and the results were consistent with the physiological and biochemical experiments.【Conclusion】The multiple PCR method is specific, stable and reliable for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae.

Key words: Vibrio , detection , multiple PCR

魏霜, 冼钰茵, 赵晖, 吴希阳. 多重PCR检测四种食源性病原弧菌[J]. 中国农业科学, 2013, 46(8): 1682-1686.

WEI Shuang, XIAN Yu-Yin, ZHAO Hui, WU Xi-Yang. Simultaneous Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae Using Multiplex PCR[J]. Scientia Agricultura Sinica, 2013, 46(8): 1682-1686.

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多重PCR检测四种食源性病原弧菌

Simultaneous Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae Using Multiplex PCR

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